Effects of type I/type II interferons and transforming growth factor-beta on B-cell differentiation and proliferation. Definition of costimulation and cytokine requirements for immunoglobulin synthesis and expression

In this report, we sought to determine the role of selected type I interferons [interferon-alpha (IFN-alpha) and interferon-tau (IFN-tau)], IFN-gamma and transforming growth factor-beta (TGF-beta) in the regulation of bovine antibody responses. B cells were stimulated via CD40 in the presence or absence of B-cell receptor (BCR) cross-linking. IFN-alpha enhanced IgM, IgG2 and IgA responses but did not enhance IgG1 responses. BCR signalling alone was more effective at inducing IgG2 responses with IFN-alpha than dual cross-linking with CD40. Recombinant ovine IFN-tau was less effective at inducing IgG2 responses when compared with IFN-alpha, though IgA responses were similar in magnitude following BCR cross-linking. At higher concentrations, IFN-tau enhanced IgA responses greater than twofold over the levels observed with IFN-alpha. Previous studies have shown that addition of IFN-gamma to BCR or pokeweed mitogen-activated bovine B cells stimulates IgG2 production. However, following CD40 stimulation alone, IFN-gamma was relatively ineffective at stimulating high-rate synthesis of any non-IgM isotype. Dual cross-linking via CD40 and the BCR resulted in decreased synthesis of IgM with a concomitant increase in IgA and similar levels of IgG2 production to those obtained via the BCR alone. We also assessed the effects of endogenous and exogenous TGF-beta on immunoglobulin synthesis by bovine B cells. Exogenous TGF-beta stimulates both IgG2 and IgA production following CD40 and BCR cross-linking in the presence of IL-2. Blocking endogenous TGF-beta did not inhibit the up-regulation of IgG2 or IgA by interferons.     

Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1

The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex. Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-28). To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed. From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains. Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs. The domain responsible for these two activities was mapped to a conserved region near the N terminus. Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs. This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast. Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo.     

Cloning and sequencing of a full-length sea bream (Sparus aurata) beta-actin cDNA

The pathogenesis of human African trypanosomiasis (HAT) has been the object of considerable research interest but has remained incompletely understood. The importance of cytokines in the pathophysiology of this protozoan infection is now widely recognized, but the full spectrum of cytokines involved has yet to be determined. In the present investigation we compared the plasma concentrations of TNF-alpha and IL-10 in normal African controls and patients suffering from advanced meningocephalic (late-stage) Trypanosomiasis brucei (T.b.) gambiense infections, before and after treatment with the arsenical trypanocide melarsoprol. We found that patients with late-stage T. b. gambiense exhibit chronically elevated circulating levels of both of these cytokines, and that these levels quickly decline following melarsoprol treatment. These findings confirm that TNF-alpha is involved in the immunopathogenesis of late-stage African trypanosomiasis and suggest that IL-10 may also play an important regulatory role in this disease.     

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: functional and localization studies

Familial apolipoprotein C-II (apo C-II) deficiency is an autosomal recessive genetic disorder characterized by fasting hypertriglyceridemia and accumulation of chylomicrons in the plasma. To elucidate the genetic defect, the apo C-II gene of a neonatal Japanese patient (C-IITokyo) was analyzed. Nucleotide sequence analysis showed a G+1 to C transversion at the donor splice site of intron 2 (INT2 G+1 to C). Restriction fragment length polymorphism analyses of the patient's family members with Hph I showed that the patient was homozygous and the parents were heterozygous for the INT2 G+1 to C mutation. Although consanguinity could not be demonstrated, haplotype analysis of the C-II gene revealed the identity of the patient's alleles on the mutation, suggesting that the parents had a common Japanese ancestor. Sequence analysis of the patient's cDNA isolated from peripheral blood lymphocytes revealed that the INT2 G+1 to C mutation causes skipping of exon 2, which encodes the initiation codon, and results in deficiency of apo C-II proteins. The outstanding feature of our patient was that he showed severe hypertriglyceridemia beginning in the neonatal period, a feature not reported in a case of apo C-II deficiency (C-IIHamburg) with the same mutation as our patient. A previous report of another case of apo C-II deficiency (C-IIToronto) suggested that the apo E4 isoform is associated with higher levels of plasma triglycerides in subjects heterozygous for the apo C-II mutation. Determination of the apo E isoform of our patient revealed that apo E4 was coinherited with the INT2 G+1 to C mutation, whereas the apo E isoform has been reported to be E2/3 in C-IIHamburg. We speculate that apo E4/4 aggravated the hypertriglyceridemia in our patient with apo C-II deficiency.     

Intergroup relations: insights from a theoretically integrative approach

In social psychology, specific research traditions, which often spring up in response to external events or social problems, tend to perpetuate the theoretical assumptions and methodological approaches with which they began. As a result, theories and methods that have proven powerful in 1 topic area are often not applied in other areas, even to conceptually similar issues. The authors adopt a theoretically integrative approach to the topic of intergroup relations. Theories and empirical approaches from the domains of attitudes, impression formation, the self, personal relationships, and norms offer many new insights into problematic issues, such as repeated findings of dissociations among stereotyping, prejudice, and discrimination. This integrative approach not only promises new theoretical advances, but also suggests numerous potential practical approaches to limiting or reducing destructive patterns of intergroup relations.     

Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy

Brain areas damaged by stroke and seizures express high levels of the 72-kd heat shock protein (HSP72). Whether HSP72 represents merely a marker of stress or plays a role in improving neuron survival in these cases has been debated. Some induced tolerance experiments have provided correlative evidence for a neuroprotective effect, and others have documented neuroprotection in the absence of HSP72 synthesis. We report that gene transfer therapy with defective herpes simplex virus vectors overexpressing hsp72 improves neuron survival against focal cerebral ischemia and systemic kainic acid administration. HSP72 overexpression improved striatal neuron survival from 62.3 to 95.4% in rats subjected to 1 hour of middle cerebral artery occlusion, and improved survival of hippocampal dentate gyrus neurons after systemic kainic acid administration, from 21.9 to 64.4%. We conclude that HSP72 may participate in processes that enhance neuron survival during transient focal cerebral ischemia and excitotoxin-induced seizures.     

Sleep-promoting effects of melatonin: at what dose, in whom, under what conditions, and by what mechanisms?

Hydrocodone is a semisynthetic opioid with analgesic and antitussive properties qualitatively similar to other opioid agonists. Ibuprofen is a nonsteroidal antiinflammatory agent with analgesic and antipyretic activity and is an effective, primarily peripheral-acting antiinflammatory analgesic. The objective of this clinical trial was to determine the additive analgesic effect of the combination of 15 mg hydrocodone bitartrate with 400 mg ibuprofen, relative to 400 mg ibuprofen alone and placebo, in the treatment of postoperative pain. The single-dose analgesic efficacy of the combination of hydrocodone bitartrate with ibuprofen was compared with ibuprofen alone and placebo in 120 patients with moderate or severe postoperative pain after abdominal surgery. Analgesia was measured during the 6-hour period after dosing based on onset of relief, hourly and summary variables, and duration of effect. A significantly greater proportion of patients treated with the hydrocodone/ibuprofen combination reported onset of relief compared with ibuprofen or placebo; however, the distribution functions for time to onset of relief did not differ among treatments. Hydrocodone with ibuprofen and ibuprofen alone were significantly more effective than placebo for all measures of analgesia. The combination of hydrocodone with ibuprofen was significantly superior to ibuprofen for all hourly analgesic evaluations, weighted sum of pain intensity differences (SPID), total pain relief (TOTPAR), and global rating of study medication. No patients in the hydrocodone with ibuprofen group required analgesic remedication during the 6-hour study period, compared with 25% and 82% in the ibuprofen and placebo groups, respectively. The analgesic superiority of 15 mg hydrocodone bitartrate combined with 400 mg ibuprofen compared with 400 mg ibuprofen alone was demonstrated across many efficacy variables.     

PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27(KIP1) and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27(KIP1) and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27(KIP1).     

Structure and activity of the hairpin ribozyme in its natural junction conformation: effect of metal ions

The natural form of the hairpin ribozyme consists of a four-way RNA junction of which the single-stranded loop-carrying helices are adjacent arms. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. We find that the junction-based form of the hairpin ribozyme is active in magnesium, calcium, or strontium ions, but not in manganese, cadmium, or sodium ions. Using fluorescence resonance energy transfer experiments, we have investigated the global structure of the ribozyme. The basic folding of the construct is based on pairwise helical stacking, so that the two loop-carrying arms are located on opposite stacked helical pairs. In the presence of magnesium, calcium, or strontium ions, the junction of the ribozyme undergoes a rotation into a distorted antiparallel geometry, creating close physical contact between the two loops. Manganese ions induce the same global folding, but no catalytic activity; this change in global conformation is therefore necessary but not sufficient for catalytic activity. Fitting the dependence of the conformation on ionic concentration to a two-state model suggests that cooperative binding of two ions is required to bring about the folding. However, further ion binding is required for cleavage activity. Cobalt hexammine ions also bring about global folding, while spermidine generates a more symmetrical form of the antiparallel structure. Cadmium ions generate a different folded form, interpreted in terms of close loop-loop association while the junction is unfolded. Sodium ions were unable to induce any folding of the ribozyme, which remained slightly parallel. These results are consistent with a folding process induced by the binding of two group IIA metal ions, distributed between the junction and the loop interface.     

Neuropathological findings in eight children with cerebro-oculo-facio-skeletal (COFS) syndrome

Cerebro-oculo-facial-skeletal (COFS) syndrome is a rare autosomal recessive disorder with microcephaly, severe mental retardation, and death in childhood. The pathogenesis is unknown. Neuropathological features of 8 children with COFS syndrome are presented. Seven of the children, ranging in age from 36 weeks gestation to 5 years 8 months, are of North American aboriginal background from Manitoba, Canada. The eight child is a 3-year-old Caucasian male. In all children there was severe microencephaly and mild ventriculomegaly. Cerebral myelination appeared to be delayed in one infantile case. Swollen ubiquitinated granular cells appeared in the white matter shortly after birth. Older children displayed cortical neuron loss, patchy or diffuse absence of myelin and gliosis in the white matter, and pericapillary and parenchymal mineralization in the globus pallidus and to a lesser extent the putamen and cerebral cortex. The cerebellum of older children exhibited severe degenerative changes involving the internal granular layer and Purkinje cell layer. The neuropathological changes, previously not well documented, suggest that COFS syndrome is associated with a degenerative process that begins in utero and affects many brain cell types. Similarities to Cockayne syndrome are discussed.